[Cytometry] Trypan Blue quenching

Iain Johnson iainjohnsonconsulting at gmail.com
Wed May 11 19:39:30 EDT 2011


Trypan Blue is not at all specific for FITC.  It works by introducing an
optical filter into the sample, which is why it has to be continuously
present and cannot be washed out.  Anti-fluorescein antibodies quench FITC
through a specific molecular interaction with the fluorophore.  The
downsides of anti-fluorescein are that it tends to be cost-prohibitive,
particularly if your sample volumes are large (despite the fact that the
working concentrations can be orders of magnitude lower than Trypan Blue due
to the specificity of the interaction).  The other downside is that the
binding affinity (and hence quenching efficiency) is somewhat dependent on
what the FITC is conjugated to.

Another alternative for these applications is to use the pH-sensitive dye
pHrodo from Molecular Probes.  Its fluorescence is relatively weak at pH 7
and increases progressively upon acidification.  Thus the background
fluorescence from extracellular dye is intrinsically low and you don't need
an additional quencher.

Iain

Iain Johnson Consulting
Eugene, OR
(541) 285-8296



On Tue, May 10, 2011 at 11:27 AM, Chris Norbury <ccn1 at psu.edu> wrote:

> We're trying to quantify uptake of exogenous materials (various kinds) by
> flow in a heterogeneous population of cells (sorting not possible for
> biosafety reasons).  Our 37 vs 4 degree controls are not that wonderful due
> to surface binding and possibly recycling, so we'd like to quench surface
> fluorescence to quantify only what is internalized.  I know a lot of folks
> don't go to these lengths to measure endocytosis/phagocytosis but we'd like
> to.
>
> We've quenched with Trypan Blue and it seems to do a good job with FITC
> labeled substrates.  BUT, it works best if present even when running,
> rather
> than as an incubation followed by washing.  This precludes the use of other
> fluorophores to identify our cell populations if Trypan Blue quenches most
> other colors/dyes.  Does anyone know of the specificity of Trypan Blue for
> FITC or of any other method of cell surface quenching that might be more
> appropriate for us to use (we've considered acid stripping).
>
> Thanks in advance for any input.
>
> Chris
>
> --
> Chris Norbury, Ph.D
> Associate Professor of Microbiology and Immunology
> Penn State Milton S. Hershey College of Medicine
> Room C6764E, Dept. of Microbiology and Immunology, H107
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>
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